Diagnosis of cowden and cowden-like syndrome by detection of decreased killin expression

ABSTRACT

A method of diagnosing Cowden syndrome (CS) and Cowden-like Syndrome (CLS) is described. The method includes diagnosing CS and CLS in a subject by identifying a decrease in expression of the KILLIN gene, or by identifying hypermethylation of the KILLIN promoter region. Kits for diagnosing CS and CLS by identifying subjects having KILLIN promoter region hypermethylation and primers specific for a methylated KILLIN promoter region are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/578,061, filed Dec. 20, 2011, which is incorporated herein byreference in its entirety.

GOVERNMENT FUNDING

The present invention was made, in part, with government support underNational Cancer Institute Grant No. P01 CA 124570. The U.S. Governmenthas certain rights in this invention.

BACKGROUND

Germline mutations of the phosphatase and tensin homolog (PTEN) gene(UCSC ID, uc001kfb.2; RefSeq, NM_000314), encoding deletions onchromosome 10, cause 25% of autosomal-dominant Cowden syndrome whichminimally occur in 1 in 200,000 live births. These mutations result in asyndrome characterized by macrocephaly and typical mucocutaneousfeatures (trichilemmomas, papillomatous papules) and hamartomas, withincreased risk of various malignancies, approximately 28% lifetime riskfor thyroid cancer, and as much as 50% lifetime risk for female breastcancer over the general population.

However, only 5% of this heterogeneous group referred to as havingCowden-like syndrome and who have some features of Cowden syndrome butdo not meet diagnostic criteria, have germline PTEN mutations. In theabsence of germline PTEN mutations, approximately 10% of individualswith Cowden syndrome or Cowden-like syndrome harbor germline succinatedehydrogenase variants SDHB and SDHD. Overall, germline PTEN mutationsand deletions and SDHx variants account for 35% of Cowden syndrome and6% to 11% of individuals with Cowden-like syndrome phenotypic features.

Cowden syndrome is a great clinical mimic and is difficult to recognizebecause every patient shows variable expression and penetrance.Importantly, many individuals in the general population share one ormore features of Cowden syndrome but may not have Cowden syndrome andmay not even harbor alterations in any predisposition genes. Many suchpatients present to primary care and other specialty clinicians who arecalled upon to recognize such individuals because individuals withspecific gene mutations have increased risks of different spectra ofneoplasias.

Somatic alterations of CpG island DNA hypermethylation and chromatinmodification have been widely documented in human cancers. Jones et al.,Cell, 128, 683-692 (2007). Regions in which CpG are exceptionallyintegrated are known as CpG islands. The CpG islands refer to siteswhich are 0.2-3 kb in length, and have a C+G content of more than 50%and a CpG ratio of more than 3.75%. There are about 45,000 CpG islandsin the human genome, and they are mostly found in promoter regionsregulating the expression of genes. In the case of genes whose mutationsare attributed to the development of cancer in congenital cancer but donot occur in acquired cancer, the germline methylation of promoter CpGislands occurs instead of mutation. Typical examples include thepromoter germline methylation of genes, such as acquired renal cancerVHL (von Hippel Lindau), breast cancer BRCA1, colorectal cancer MLH1,and stomach cancer E-CAD.

DNA methylation changes are not only detectable in tumors, but also inblood, as tumor-derived DNA is released into the bloodstream due totumor necrosis and apoptosis. Cancer-specific DNA methylationalterations present in cancer tissues and blood of cancer patients canserve as diagnostic markers for risk assessment, progression, earlydetection, treatment prediction and monitoring. Laird, P. W., Nat RevCancer, 3, 253-266 (2003).

In the context of a difficult-to recognize syndrome, identification ofadditional cancer predisposition genes would facilitate moleculardiagnosis, genotype-specific predictive testing of family members whoare as yet clinically unaffected, genetic counseling, and medicalmanagement. Relevant to primary care, once a mutation or alteration isfound, primary care physicians must have a basic understanding ofgene-specific cancer risks as they do play and will increase their roleas coordinators of gene-specific personalized management, surveillance,and other related factors of care.

SUMMARY

PTEN is a well-characterized tumor suppressor phosphatase involved incellular regulation via G1 cell cycle arrest and apoptosis. Salmena etal., Cell 133(3): 403-414 (2008). Interestingly, a novel gene, KILLIN(UCSC, uc009xti.2; RefSeq, NM_001126049), which also resides in the10q23.31 chromosomal region, is involved in cell cycle arrest and isregulated by TP53, similar to PTEN. PTEN and KILLIN share the sametranscription start site but are transcribed in opposite directions.KILLIN has been shown to be necessary and sufficient for TP53-inducedapoptosis. Cho et al., Proc Natl Acad Sci USA., 105(14): 5396-5401(2008). This high-affinity DNA binding protein inhibits eukaryotic DNAsynthesis in vitro and causes S phase arrest before apoptosis in vivo.Because of similar function to PTEN, KILLIN was investigated as apredisposition gene in patients with Cowden syndrome or Cowden-likesyndrome.

Epigenetic alterations play an important role in cancer progressionthrough hypermethylation and silencing of tumor suppressor genes, andsomatic PTEN hypermethylation has been recognized as a means of PTENdown-regulation in a subset of malignancies. When promoter CpG islandsare methylated, the reason why the expression of the corresponding genesis blocked is not clearly established, but is presumed to be because amethyl CpG-binding protein or a methyl CpG-binding domain protein, andhistone deacetylase, bind to methylated cytosine, thereby causing achange in the chromatin structure of chromosomes and a change in histoneprotein.

The inventors sought to address the hypothesis that germline methylationof the 10q23.31 bidirectional promoter CpG island (a region of at least200 base pairs [bp] with a GC content of ≧50% and an observed andexpected CpG ratio of >60%) silences PTEN, KILLIN, or both. This,consequently, would account for patients with Cowden syndrome orCowden-like syndrome features but without germline PTEN mutations ordeletions.

Accordingly, in one aspect, a method of diagnosing Cowden syndrome andCowden-like Syndrome is provided. The method includes the steps ofobtaining a biological sample from a subject; determining the level ofexpression of the KILLIN gene in the biological sample; and comparingthe level of expression of the KILLIN gene in the biological sample to acontrol value for KILLIN gene expression; wherein a lower level ofexpression of the KILLIN gene in the biological sample relative to theKILLIN expression control value provides a diagnosis that the subjecthas a substantially increased risk of having Cowden syndrome orCowden-like syndrome.

In one embodiment, the level of KILLIN gene expression is measured bydetecting the methylation of a KILLIN promoter region, whereinhypermethylation of the KILLIN promoter region indicates a lower levelof KILLIN gene expression. In a further embodiment, the step ofdetecting methylation of the KILLIN promoter region includes the step ofbringing the KILLIN promoter region into contact with sodium bisulfateunder conditions suitable to modify unmethylated cytosine of the KILLINpromoter region into uracil. In a further embodiment, sodium bisulfateis used as part of a combined bisulfate restriction analysis.

In additional embodiments, the subject already has one or more symptomsof Cowden syndrome, such as breast cancer, thyroid cancer, or kidneycancer. In yet further embodiments, the method also includes the step ofproviding a therapeutic intervention for a subject identified as havinga substantially increased risk of having Cowden syndrome or Cowden-likesyndrome.

In another aspect, a kit for diagnosing Cowden Syndrome and Cowden-likeSyndrome is provided. The kit may vary depending on the method used todetect DNA methylation. In one embodiment, the kit includes a carriercompartmentalized to include a plurality of containers and to receive aDNA sample including the KILLIN promoter region from a subject therein.The carrier includes a first container including sodium bisulfate, andthe solvents and reagents necessary to selectively convert unmethylatedcytosine of the DNA sample into uracil; a second container containing aPCR primer pair corresponding to the methylated base sequence of theKILLIN promoter region and the solvents and reagents necessary to obtainan amplified base sequence; and a third container containing a labeledprobe complementary to the amplified base sequence. The kit alsoincludes means for detecting the labeled probe to quantitatively analyzethe amount of methylation of the KILLIN promoter region; and a KILLINpromoter region control.

In a further embodiment, the kit includes instructions for use of thekit to compare the amount of methylation of the KILLIN promoter regionin the DNA sample to a KILLIN promoter region control. Hypermethylationof the KILLIN promoter region indicates a diagnosis of Cowden syndromeor Cowden-like Syndrome for the subject. The kit can use a PCR primerpair is selected from the group consisting of BS PCR forward primer SEQID NO: 12 and BS PCR reverse primer SEQ ID NO: 13. This PCR primer pairprovides primers for DNA methylation analysis using the combinedbisulfate restriction analysis method.

Another aspect provides a method for treating a subject having Cowdensyndrome or Cowden-like syndrome by administering to the subject atherapeutically effective amount of a DNA methyltransferase inhibitor.In a further embodiment, the method also includes administering ahistone deacetylase inhibitor to the subject. This method can be usefulfor a subject that has been found to lack germline PTEN mutation, sincethis indicates that it is more likely that the Cowden syndrome is aresult of methylation of the KILLIN promoter region.

Additional features and advantages of the exemplary embodiments will beset forth in part in the description that follows, and in part will beobvious from the description, or may be learned by practice of theexemplary embodiments disclosed herein. The objects and advantages ofthe exemplary embodiments disclosed herein will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims. It is to be understood that both the foregoingbrief summary and the following detailed description are exemplary andexplanatory only and are not restrictive of the embodiments disclosedherein or as claimed.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate some embodiments disclosedherein, and together with the description, serve to explain principlesof the exemplary embodiments disclosed herein.

FIG. 1 provides a sequence for the adjacent PTEN and KILLIN genes onchromosome 10. The sequence includes the PTEN gene (SEQ ID NO: 1), theKILLIN gene (SEQ ID NO: 2), and the KILLIN promoter region (SEQ ID NO:3). The individual sequences are designated using square brackets. ThePTEN and KILLIN genes are transcribed in opposite directions, as shownin FIG. 2. The KILLIN promoter region is embedded within the PTEN gene.

FIG. 2 provides a schematic of the Genomic Structure of the KILLIN andPTEN Genes on Chromosome 10. The region analyzed for DNA methylation isindicated, with the numbers showing the location of the bisulfitepolymerase chain reaction product with respect to the translation startsite (mRNA [messenger RNA]) of the PTEN gene. As depicted, the KILLINpromoter overlaps with the 5′UTR (untranslated region) and coding regionof PTEN. bp indicates base pair.

FIG. 3 provides experimental results and a pictoral representation ofgermline DNA methylation of PTEN and KILLIN in Cowden Syndrome andCowden-like Syndrome. FIG. 3A, Example of combined bisulfite restrictionanalysis (COBRA) of polymerase chain reaction (PCR) products from asubset of patients with Cowden syndrome or Cowden-like syndrome (numbersrefer to patient numbers). The 0% and 100% are from peripheral bloodDNA, the 100% having been in vitro methylated with Sss I methylase.These serve as negative and positive controls for methylation patternanalysis of the patients. An increase in the intensity of smallerdigested bands compared with the control samples indicates increasedmethylation in the tumor DNA. FIG. 3B, Results from bisulfite sequencinganalysis of 8 of the patient samples. Each row of circles represents anindividual cell clone. bp indicates base pair.

FIG. 4 provides graphs showing the results of quantitative mRNA analysisof PTEN and KILLIN Expression with germline methylation in controls andpatients With Cowden Syndrome or Cowden-like Syndrome. Quantitativereverse transcription polymerase chain reaction analysis of fourcontrols and eight patient samples. All samples were first normalized totheir own internal control (GAPDH). The average of the controls, set to1, was used for normalization for all samples. The top panel displaysthe expression for PTEN, which reveals significantly increasedexpression (at the P<0.05 threshold) in 3 patient samples (patients 21,446, and 1350) compared with the controls, while only 1 sample showedsignificantly decreased expression (patient 397). The bottom panelreveals significantly decreased KILLIN expression in all patient samplesanalyzed. Error bars indicate 95% confidence intervals; mRNA, messengerRNA.

FIG. 5 provides graphs showing the results of quantitative mRNA analysisof PTEN and KILLIN expression with germline methylation, with andwithout demethylation and histone deacetlyase inhibition treatment.Quantitative reverse transcription polymerase chain reaction analysiswas performed on the complementary DNA from cells with (+) and without(−) drug exposure (see “Methods” section) to detect changes inexpression from the demethylation and histone deacetylase inhibitiontreatment. All values were first normalized to their internal control(GAPDH). The fold increase or decrease in expression in the drug-treatedsamples is derived by normalizing to its untreated counterpart, whichwas set as 1. PTEN expression is shown on the top panel and reveals asignificant decrease in PTEN expression following demethylation in allbut 1 cell line. Patient 397 that showed an increase in PTEN expressionfollowing demethylation treatment alone was not significant (P=0.42).The bottom panel shows KILLIN expression following demethylation and/orinhibition of histone deacetylation, which shows a significant increasein expression in 7 of 8 cell lines (patient 446 was the exception). mRNAindicates messenger RNA. Error bars indicate 95% confidence intervals.

FIG. 6 provides a schematic showing the effect of methylation ontranscription of KILLIN and PTEN. The model depicts where the observedmethylation resides with respect to both KILLIN and PTEN and shows theregions where TP53 binds for PTEN and is blocked from binding for KILLINtranscriptional activation. mRNA indicates messenger RNA; UTR,untranslated region; bp, base pair.

FIG. 7 provides a graph showing the results of chromatinimmunoprecipitation analysis. Chromatin immunoprecipitation analysis ofTP53 pulldown of either KILLIN's or PTEN's TP53 binding element incontrols and Cowden syndrome patients. All samples are normalized totheir negative control, IgG. Varied enrichment of the KILLIN and PTENTP53 binding sites was observed in the control samples, whereas asignificantly greater amount of region 1 (PTEN's TP53 binding site) waspulled down in 3 of 4 patient cell lines (patient 31 was the exception).Error bars indicate 95% confidence intervals.

FIG. 8 provides a graph showing a comparison of transcriptionalactivation following methylation of the same promoter sequence in KILLINand PTEN. In vitro methylation with Sss I methylase was performed forboth the PTEN and KILLIN luciferase promoter constructs. The constructscontained the same promoter sequence (either in orientation for KILLINor in the opposite orientation for PTEN), which includes 1 to 1344 basepair of sequence upstream of the translation start site of PTEN.Luciferase promoter analysis of PTEN and KILLIN promoter activity wasdone using the MDA-MB-453 breast cancer cells in the absence (wild-type[WT]) or presence (+TP53) of TP53 transfection. All values were firstnormalized to their internal control, Renilla luciferase. The foldincrease in the samples with TP53 overexpression was attained bynormalization to those without TP53 transfection, which was set as 1.The PTEN constructs showed significant activation by TP53 regardless ofmethylation status (P=0.01). However, the KILLIN-methylated constructshowed significantly less activation by TP53 compared with theunmethylated KILLIN luciferase construct (P=0.008). The WT analysis wasnot significant (P=0.78). Error bars indicate 95% confidence intervals.

DETAILED DESCRIPTION

The exemplary embodiments disclosed herein will now be described byreference to some more detailed exemplary embodiments, with occasionalreference to the accompanying figures. These exemplary embodiments may,however, be embodied in different forms and should not be construed aslimited to the embodiments set forth herein. Rather, these exemplaryembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the exemplary embodimentsto those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these exemplary embodiments belong. The terminologyused in the description herein is for describing particular exemplaryembodiments only and is not intended to be limiting of the exemplaryembodiments. As used in the specification and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present embodiments. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldbe construed in light of the number of significant digits and ordinaryrounding approaches.

As used herein, the term “diagnosis” can encompass determining thepresence and nature of disease or condition in a subject. “Diagnosis”can also encompass diagnosis in the context of rational therapy, inwhich the diagnosis guides therapy, including initial selection oftherapy, modification of therapy (e.g., adjustment of dose and/or dosageregimen), and the like. Diagnosis does not imply certainty with regardto the nature of the disease or condition identified, but rather thesubstantial likelihood that the disease or condition is present. Forexample, a subject diagnosed as having Cowden syndrome may be 10× or100× more likely to have Cowden syndrome relative to a subject that hasnot been diagnosed as having Cowden syndrome.

Diagnosis may be useful for early detection of a disease. As usedherein, the term “early detection” of a disease (e.g., Cowden Syndromeand its associated cancers) refers to discovering the likelihood ofcancer prior to metastasis, and preferably before observation of amorphological change in a tissue or cell. Furthermore, the term “earlydetection” of cell transformation refers to the high probability of acell to undergo transformation in its early stages before the cell ismorphologically designated as being transformed.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, refers to a species of mammal, including, butnot limited to, primates, including simians and humans, equines (e.g.,horses), canines (e.g., dogs), felines, various domesticated livestock(e.g., ungulates, such as swine, pigs, goats, sheep, and the like), aswell as domesticated pets and animals maintained in zoos.

As used herein, the term “therapeutically effective amount” is meant anamount of a compound effective to yield the desired therapeuticresponse. For example, an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, or to shrink the cancer orprevent metastasis. The therapeutically effective amount will vary withsuch factors as the particular condition being treated, the physicalcondition of the patient, the type of mammal or animal being treated,the duration of the treatment, the nature of concurrent therapy (ifany), and the specific formulations employed and the structure of thecompounds or its derivatives.

As used herein, “proliferative growth disorder, “neoplastic disease,”“tumor; “cancer” are used interchangeably as used herein refers to acondition characterized by uncontrolled, abnormal growth of cells.Examples of cancer include but are not limited to, carcinoma, blastoma,and sarcoma. As used herein, the term “carcinoma” refers to a new growththat arises from epithelium, found in skin or, more commonly, the liningof body organs.

The term “in need of such treatment” as used herein refers to a judgmentmade by a care giver such as a physician, nurse, or nurse practitionerin the case of humans that a patient requires or would benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a care giver's expertise, but that include the knowledgethat the patient is ill, or will be ill, as the result of a conditionthat is treatable by the compounds of the invention.

A method of diagnosing Cowden Syndrome (CS) and Cowden-like Syndrome(CLS) is described herein. Cowden syndrome, which is also known as“Cowden's disease,” and “Multiple hamartoma syndrome,” is a rareautosomal dominant inherited disorder characterized by multipletumor-like growths called hamartomas and an increased risk of certainforms of cancer. Cowden syndrome is associated with loss-of-functionmutations in the tumor suppressor gene PTEN, leading to hyperactivity ofthe mTOR pathway. These mutations lead to characteristic featuresincluding macrocephaly, intestinal hamartomatous polyps, benign skintumors (multiple trichilemmomas, papillomatous papules, and acralkeratoses) and dysplastic gangliocytoma of the cerebellum(Lhermitte-Duclos disease). In addition, there is a pre-disposition tobreast carcinoma, carcinoma of the thyroid, endometrial carcinoma, renalcell carcinoma, colorectal carcinoma and melanoma. Cowden-like syndromeis used to describe the disorder in which patients have many of thefeatures of Cowden syndrome, but do not meet the formal diagnosticcriteria.

In one embodiment, the method of diagnosing CS or CLS includes the stepsof: (a) obtaining a biological sample from a subject; (b) determiningthe level of expression of the KILLIN (KLLN) gene in the biologicalsample; and (c) comparing the level of expression of the KILLIN gene inthe biological sample to a control value for KILLIN gene expression. Alower level of expression of the KILLIN gene in the biological samplerelative to the KILLIN expression control value provides a diagnosisthat the subject has a substantially increased risk of having Cowdensyndrome or Cowden-like syndrome.

Biological Samples

Biological samples include tissue samples (e.g., a portion of an organ),a cell sample (e.g., peripheral leukocytes) and biological fluids suchas urine and blood-related samples (e.g., whole blood, serum, plasma,and other blood-derived samples), urine, cerebral spinal fluid,bronchoalveolar lavage, and the like. Methods of obtaining samplesand/or extracting nucleic acid or protein from such samples aredescribed herein and known to those skilled in the art.

A biological sample may be fresh or stored (e.g. blood or blood fractionstored in a blood bank). Samples can be stored for varying amounts oftime, such as being stored for an hour, a day, a week, a month, or morethan a month. The biological sample may be a biological fluid expresslyobtained for the assays of this invention or a biological fluid obtainedfor another purpose which can be subsampled for the assays of thisinvention.

As used herein, the term “expression level of the KILLIN gene” refers tothe amount of mRNA transcribed from the KILLIN gene that is present in abiological sample. The expression level can be detected with or withoutcomparison to a level from a control sample or a level expected of acontrol sample. The expression level can be determined by measuring theamount of mRNA, or by measuring the amount of protein formed from themRNA. The expression level of the KILLIN gene may be decreased by atleast about 2 fold, at least about 5 fold, at least about 10 fold, atleast about 20 fold, or at least about 50 fold.

A variety of methods may be used to determine the level of expression ofthe KILLIN gene. For example, the level of expression of the KILLIN genecan be obtained by determining the relative levels of mRNA beingexpressed using quantitative real-time polymerase chain reaction (qPCR).A key feature of qPCR is that the amplified DNA is detected as thereaction progresses in real time. This differs from standard PCR, wherethe product of the reaction is detected at its end. Two common methodsfor detection of products in real-time PCR are non-specific fluorescentdyes that intercalate with any double-stranded DNA, andsequence-specific DNA probes consisting of oligonucleotides that arelabeled with a fluorescent reporter which permits detection only afterhybridization of the probe with its complementary DNA target. SeeVanGuilder et al., Biotechniques 44 (5): 619-626 (2008).

Another method of determining the level of KILLIN gene expression is topurify the expressed Killin protein and directly determine its level ofexpression. Methods for purifying the Killin protein are described inU.S. Pat. No. 7,576,191, the disclosure of which is incorporated hereinby reference. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified and/or quantified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areimmunohistochemistry, ion-exchange chromatography, exclusionchromatography; polyacrylamide gel electrophoresis; isoelectricfocusing. A particularly efficient method of purifying peptides is fastprotein liquid chromatography or even HPLC.

Another embodiment of the method of diagnosing CS or CLS determines thelevel of KILLIN gene expression by detecting the level of methylation ofthe KILLIN promoter region. Hypermethylation of the KILLIN promoterregion corresponds to a decreased level of KILLIN gene expression. Asused herein, the term “hypermethylation” refers to the methylation statecorresponding to an increased presence of 5-methyl-cytosine (“5-mCyt”)at one or a plurality of CpG dinucleotides within a DNA sequence of atest DNA sample, relative to the amount of 5-mCyt found at correspondingCpG dinucleotides within a normal control DNA sample. In particular,hypermethylation refers to the methylation of a CpG island. As describedherein, methylation of the KILLIN promoter results in decreasedformation of the KILLIN promoter, resulting in a lower level of KILLINexpression.

The method of diagnosing CS or CLS includes the steps of: (a) obtaininga biological sample including DNA from a subject; (b) determining thelevel of methylation of the KILLIN promoter region in the biologicalsample; and (c) comparing the level of methylation of the KILLINpromoter region in the biological sample to a control value for KILLINpromoter expression. A higher level of methylation of the KILLINpromoter region in the biological sample relative to methylation of theKILLIN promoter region control value provides a diagnosis that thesubject has a substantially increased risk of having Cowden syndrome orCowden-like syndrome.

The method of diagnosing Cowden syndrome or Cowden-like syndromeincludes the step of obtaining a biological sample including DNA from asubject. More specifically, the DNA is a DNA that includes the KILLINpromoter region (SEQ ID NO: 3). The biological sample including DNA caninclude any of the biological samples described herein. However, in someembodiments, it may be preferable to obtain the biological sample fromtissue that has been characterized as being cancerous or precancerous.DNA (deoxyribonucleic acid), as is understood by those skilled in theart, is a molecule consisting of two long polymers of simple unitscalled nucleotides with a backbone made of alternating sugars(deoxyribose) and phosphate groups that forms a double-stranded helix.The nucleotides include guanine, adenine, thymine, and cytosine, whichare referenced using the letters G, A, T, and C. The term “nucleotidesequence,” as used herein, refers to an oligonucleotide, nucleotide, orpolynucleotide of single-stranded or double stranded DNA, or fragmentsthereof.

The KILLIN promoter is found within the KILLIN promoter region. Theexact nucleotide sequence corresponding to the KILLIN promoter has notyet been identified, but it is known that a sequence within the KILLINpromoter region expresses the KILLIN promoter. The KILLIN promoterregion may be part of a mixture of nucleic acids. The KILLIN promoterregion may be a fraction of a larger molecule or can be presentinitially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence be present in a pure form; the nucleic acid may be a minorfraction of a complex mixture, such as contained in whole human DNA.Nucleic acids contained in a sample used for detection of methylated CpGislands may be extracted by a variety of techniques such as thatdescribed by Sambrook, et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989).

DNA methylation is essential for normal development and is associatedwith a number of key processes including genomic imprinting,X-chromosome inactivation, suppression of repetitive elements, andcarcinogenesis. Between 60% and 90% of all CpGs are methylated inmammals. Unmethylated CpGs are often grouped in clusters called CpGislands, which are present in the 5′ regulatory regions of many genes.As used herein, the term “methylation” refers to the covalent attachmentof a methyl group at the C5-position of the nucleotide base cytosinewithin the CpG dinucleotides of gene regulatory region. The term“methylation state” or “methylation status” or “methylation level” or“the degree of methylation” refers to the presence or absence of 5-mCytat one or a plurality of CpG dinucleotides within a DNA sequence.

The present invention further encompasses the use of nucleotidesequences which are at least 85%, or at least 90%, or more preferablyequal to or greater than 95% identical to the KILLIN promoter region(SEQ ID NO: 3). Sequence identity as used herein refers to theproportion of base matches between two nucleic acid sequences or theproportion amino acid matches between two amino acid sequences. Whensequence homology is expressed as a percentage, e.g., 50%, thepercentage denotes the proportion of matches over the length of sequencefrom one sequence that is compared to some other sequence.

Controls

Control values are based upon the level of KILLIN expression ormethylation of the KILLIN promoter region in comparable samples obtainedfrom a reference cohort. In certain embodiments, the reference cohort isthe general population. For example, the reference cohort can be aselect population of human subjects. In certain embodiments, thereference cohort is comprised of individuals who have not previously hadany signs or symptoms indicating the presence of Cowden syndrome orCowden-like syndrome.

The control value can take a variety of forms. The control value can bea single cut-off value, such as a median or mean. Control values for thelevel of KILLIN expression or the methylation of the KILLIN promoterregion in biological samples obtained, such as for example, mean levels,median levels, or “cut-off” levels, are established by assaying a largesample of individuals in the general population or the select populationand using a statistical model such as the predictive value method forselecting a positivity criterion or receiver operator characteristiccurve that defines optimum specificity (highest true negative rate) andsensitivity (highest true positive rate) as described in Knapp, R. G.,and Miller, M. C. (1992). Clinical Epidemiology and Biostatistics.William and Wilkins, Harual Publishing Co. Malvern, Pa., which isspecifically incorporated herein by reference.

Method for Detection of Gene Methylation

The method of diagnosing Cowden Syndrome or Cowden-like Syndrome caninclude determining the level of methylation of the KILLIN promoterregion in a biological sample. A wide variety of methods are availablefor determining gene methylation, such as methylation of the KILLINpromoter region. A number of these methods are described herein.

Detection of Differential Methylation-Methylation-Specific PCR

When genomic DNA is treated with bisulfite, cytosine in the 5′-CpG′-3region remains intact, if it was methylated, but the cytosine changes touracil, if it was unmethylated. Accordingly, the method of diagnosis caninclude the step of detecting methylation of the KILLIN promoter regionincludes the step of bringing the KILLIN promoter region into contactwith sodium bisulfate under conditions suitable to modify unmethylatedcytosine of the KILLIN promoter region into uracil. Based on the basesequence converted after bisulfite treatment, PCR primer setscorresponding to a region having the 5′-CpG-3′ base sequence areconstructed. Herein, the constructed primer sets are two kinds of primersets: a primer set corresponding to the methylated base sequence, and aprimer set corresponding to the unmethylated base sequence. When genomicDNA is converted with bisulfite and then amplified by PCR using theabove two kinds of primer sets, the PCR product is detected in the PCRmixture employing the primers corresponding to the methylated basesequence, if the genomic DNA was methylated, but the genomic DNA isdetected in the PCR mixture employing the primers corresponding to theunmethylated, if the genomic DNA was unmethylated. This methylation canbe quantitatively analyzed by agarose gel electrophoresis.

Detection of Differential Methylation—Real-Time Methylation Specific PCR

Real-time methylation-specific PCR is a real-time measurement methodmodified from the methylation-specific PCR method and includes treatinggenomic DNA with bisulfite, designing PCR primers corresponding to themethylated base sequence, and performing real-time PCR using theprimers. Methods of detecting the methylation of the genomic DNA includetwo methods: a method of detection using a TanMan probe complementary tothe amplified base sequence; and a method of detection using SYBR green(an asymmetrical cyanine dye used as a nucleic acid stain). Real-timemethylation-specific PCR allows selective quantitative analysis ofmethylated DNA. A standard curve is plotted using an in vitro methylatedDNA sample, and a gene containing no 5′-CpG-3′ sequence in the basesequence is also amplified as a negative control group forstandardization to quantitatively analyze the degree of methylation.

Detection of Differential Methylation—Bisulfate Sequencing Method

Another method for detecting a methylated CpG-containing nucleic acidcomprises the steps of: bringing a nucleic acid-containing sample intocontact with an agent that modifies unmethylated cytosine; andamplifying the CpG-containing nucleic acid in the sample usingCpG-specific oligonucleotide primers, wherein the oligonucleotideprimers distinguish between modified methylated nucleic acid andnon-methylated nucleic acid and detect the methylated nucleic acid. Theamplification step is optional and desirable, but not essential. Themethod relies on the PCR reaction to distinguish between modified (e.g.,chemically modified) methylated DNA and unmethylated DNA. Such methodsare described in U.S. Pat. No. 5,786,146 relating to bisulfitesequencing for detection of methylated nucleic acid. This method is alsoreferred to herein as combined bisulfate restriction analysis.

Examples of primers suitable for combined bisulfate restriction analysisare described in example 1. Other primers suitable for combinedbisulfate restriction analysis of the KILLEN promoter region include anative sequence forward primer AAGGGAAGGTGGAAG (SEQ ID NO: 14); a nativesequence reverse primer GGCACATCCAGGGACC (SEQ ID NO: 15); a forwardprimer for bisulfate-treated DNA AAGGGAAGGTGGAAG (SEQ ID NO: 16); and areverse primer for bisulfate-treated DNA GGTATATTTAGGGATT (SEQ ID NO:17).

Detection of Differential Methylation—Pyrosequencing

The pyrosequencing method is a quantitative real-time sequencing methodmodified from the bisulfite sequencing method. Similarly to bisulfitesequencing, genomic DNA is converted by bisulfite treatment, and then,PCR primers corresponding to a region containing no 5′-CpG-3′ basesequence are constructed. Specifically, the genomic DNA is treated withbisulfite, amplified using the PCR primers, and then subjected toreal-time base sequence analysis using a sequencing primer. The degreeof methylation is expressed as a methylation index by analyzing theamounts of cytosine and thymine in the 5′-CpG-3′ region.

Detection of Differential Methylation—PCR Using Methylated DNA-SpecificBinding Protein, Quantitative PCR, and DNA Chip Assay

When a protein binding specifically to methylated DNA is mixed with DNA,the protein binds specifically only to the methylated DNA. Thus, eitherPCR using a methylation-specific binding protein or a DNA chip assayallows selective isolation of only methylated DNA. Genomic DNA is mixedwith a methylation-specific binding protein, and then only methylatedDNA was selectively isolated. The isolated DNA is amplified using PCRprimers corresponding to the promoter region, and then methylation ofthe DNA is measured by agarose gel electrophoresis.

In addition, methylation of DNA can also be measured by a quantitativePCR method, and methylated DNA isolated with a methylated DNA-specificbinding protein can be labeled with a fluorescent probe and hybridizedto a DNA chip containing complementary probes, thereby measuringmethylation of the DNA. Herein, the methylated DNA-specific bindingprotein may be, but not limited to, McrBt.

Detection of Differential Methylation—Methylation-Sensitive RestrictionEnzyme

Detection of differential methylation can be accomplished by bringing anucleic acid sample into contact with a methylation-sensitiverestriction endonuclease that cleaves only unmethylated CpG sites. In aseparate reaction, the sample is further brought into contact with anisoschizomer of the methylation-sensitive restriction enzyme thatcleaves both methylated and unmethylated CpG-sites, thereby cleaving themethylated nucleic acid. Specific primers are added to the nucleic acidsample, and the nucleic acid is amplified by any conventional method.The presence of an amplified product in the sample treated with themethylation-sensitive restriction enzyme but absence of an amplifiedproduct in the sample treated with the isoschizomer of themethylation-sensitive restriction enzyme indicates that methylation hasoccurred at the nucleic acid region assayed. However, the absence of anamplified product in the sample treated with the methylation-sensitiverestriction enzyme together with the absence of an amplified product inthe sample treated with the isoschizomer of the methylation-sensitiverestriction enzyme indicates that no methylation has occurred at thenucleic acid region assayed.

As used herein, the term “methylation-sensitive restriction enzyme”refers to a restriction enzyme (e.g., SmaI) that includes CG as part ofits recognition site and has activity when the C is methylated ascompared to when the C is not methylated. Non-limiting examples ofmethylation-sensitive restriction enzymes include MspI, HpaII, BssHII, BstUI and NotI. Such enzymes can be used alone or in combination.Examples of other methylation-sensitive restriction enzymes include, butare not limited to SacII and EagI. The isoschizomer of themethylation-sensitive restriction enzyme is a restriction enzyme thatrecognizes the same recognition site as the methylation-sensitiverestriction enzyme but cleaves both methylated and unmethylated CGs. Anexample thereof includes MspI.

Primers for use in the methods for detecting DNA methylation aredesigned to be “substantially” complementary to each strand of the locusto be amplified and include the appropriate G or C nucleotides. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands under polymerization reaction conditions.The primers are used in the amplification process, which is an enzymaticchain reaction (e.g., PCR) in which that a target locus exponentiallyincreases through a number of reaction steps. Typically, one primer ishomologous with the negative (−) strand of the locus (antisense primer),and the other primer is homologous with the positive (+) strand (senseprimer). After the primers have been annealed to denatured nucleic acid,the nucleic acid chain is extended by an enzyme such as DNA Polymerase I(Klenow), and reactants such as nucleotides, and, as a result, + and −strands containing the target locus sequence are newly synthesized. Whenthe newly synthesized target locus is used as a template and subjectedto repeated cycles of denaturing, primer annealing, and extension,exponential synthesis of the target locus sequence occurs. The resultingreaction product is a discrete nucleic acid duplex with terminicorresponding to the ends of specific primers employed. For example, inone embodiment of the invention, the PCR primers used selected from thegroup consisting of SEQ ID NO: 12 and SEQ ID NO: 13. In particular,these PCR primers are suitable for DNA methylation analysis using thecombined bisulfate restriction analysis method.

The amplification reaction is the polymerase chain reaction (PCR) whichis well known and commonly used in the art. However, alternative methodssuch as real-time PCR or linear amplification using isothermal enzymemay also be used. In addition, multiplex amplification reactions mayalso be used.

Symptoms of Cowden Syndrome

In some embodiments of the method of diagnosis, the subject has one ormore symptoms of Cowden syndrome. For example, breast cancer, thyroidcancer, and kidney cancer are all symptoms of Cowden syndrome. Clinicalfeatures of Cowden syndrome are diverse, including breast, endometrial,thyroid, kidney and colorectal cancers, dermatologic features such asoral and skin papillomas, trichilemmomas, gastrointestinal features suchas mixed polyposis including hamartomas, and neurologic features such asautism and Lhermitte Duclos disease. Diagnostic criteria have evolvedover the years; the most recent is the Cleveland Clinic scoring system.Tan et al., American Journal of Human Genetics 88 (1): 42-56 (2011). Foran individual patient, these features may be evaluated by the ClevelandClinic web calculator to derive an individual probability of a relevantgene mutation.

The characteristic hamartomas of Cowden syndrome are small, noncancerousgrowths that are most commonly found on the skin and mucous membranes(such as the lining of the mouth and nose), but can also occur in theintestinal tract and other parts of the body. They are largely benign.However, people with Cowden syndrome have an increased risk ofdeveloping several types of cancer, including cancers of the breast,thyroid, and uterus.

Up to 75% have benign breast conditions such as ductal hyperplasia,intraductal papillomatosis, adenosis, lobular atrophy, fibroadenomas,and fibrocystic changes. Nonmedullary thyroid cancer develops in up to10 percent of affected individuals. In addition, over one-half of thoseaffected have follicular adenomas or multinodular goiter of the thyroid.Other malignancies that appear to be associated with Cowden andCowden-like syndrome include endometrial and renal cancers. Other signsand symptoms of Cowden syndrome can include an enlarged head, a rarenoncancerous brain tumor called Lhermitte-Duclos disease, and glycogenicacanthosis of the esophagus. The majority of affected individualsdevelop the characteristic skin lesions by age 20.

Kits

A kit for diagnosing Cowden syndrome and Cowden-like syndrome is alsodescribed. The kit includes a carrier compartmentalized to include aplurality of containers and to receive a DNA sample including the KILLINpromoter region from a subject therein. The kit includes a firstcontainer including sodium bisulfate, and the solvents and reagentsnecessary to selectively convert unmethylated cytosine of the DNA sampleinto uracil; a second container containing a PCR primer paircorresponding to the methylated base sequence of a KILLIN promoterregion and the solvents and reagents necessary to obtain an amplifiedbase sequence; and a third container containing a labeled probecomplementary to the amplified base sequence. The kit also includesmeans for detecting the labeled probe to quantitatively analyze theamount of methylation of the KILLIN promoter region; and a KILLINpromoter region control.

The kit includes a carrier suited for containing one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Inview of the description provided herein of the inventive method, thoseof skill in the art can readily determine the apportionment of thenecessary reagents among the container means. The solvents and reagentsnecessary to convert unmethylated cytosine into uracil and to conductPCR amplification of a methylated base sequence are known to thoseskilled in the art, and are referenced herein.

In other embodiments, kits designed to detect methylation of the KILLINpromoter region using DNA methylation detection methods other thanbisulfate sequencing methods can be used. Examples of other DNAmethylation detection include any of the DNA methylation detectionmethods described herein, such as pyro sequencing and cleavage ofunmethylated CpG sizes using a methylation-sensitive restriction enzyme.For all of these kits, if a higher level of methylation of the KILLINpromoter region is found relative to that present in controls, or theKILLIN promoter region is found to be hypermethylated, this indicatesthat a subject evaluated using the kit can be diagnosed as having Cowdensyndrome or Cowden-like syndrome.

An additional embodiment provides a kit for diagnosing CS or CLS using amethylation-sensitive restriction enzyme. In this embodiment, the kitincludes carrier means compartmentalized to receive a sample therein;and one or more containers including a first container containing areagent that sensitively cleaves unmethylated cytosine, a secondcontainer containing a PCR primer pair corresponding to the methylatedbase sequence of a KILLIN promoter region and the solvents and reagentsnecessary to obtain an amplified base sequence, and a third containing ameans for detecting the presence of a cleaved or uncleaved nucleic acid.For example, one of the container means can include a containercontaining a methylation-sensitive restriction enzyme. One or morecontainer means can also include a primer complementary to the KILLINpromoter region. In addition, one or more container means can alsocontain an isoschizomer of the methylation sensitive restriction enzyme.

In a further embodiment, a kit for diagnosing CS or CLS usingpyrosequencing is provided. In this embodiment, the kit includes carriermeans compartmentalized to receive a sample therein; and one or morecontainers including a first container containing sodium bisulfate, andthe solvents and reagents necessary to selectively convert unmethylatedcytosine of the DNA sample into uracil, and a second containercontaining a PCR primer pair corresponding to the methylated basesequence of a KILLIN promoter region and the solvents and reagentsnecessary to obtain an amplified base sequence. An additional containercan be provided to conduct real-time base sequence analysis using asequence primer, or this analysis can be conducted outside of the kit.In this embodiment, the degree of methylation is expressed as amethylation index by analyzing the amount of cytosine and thymine in the5′-CpG-3′ region of the KILLIN promoter region.

Primers contemplated for use in accordance with the present inventioninclude any primers suitable for PCR amplification of the KILLINpromoter region. The primers can include a pair of PCR primer sequencesfor DNA methylation analysis that include a forward PCR primer and areverse PCR primer, wherein the primers include from 20 to 25nucleotides and are effective to amplify SEQ ID NO: 3 using thepolymerase chain reaction. For example, the PCR primer pairs can be SEQID NO: 12 (the forward primer) and SEQ ID NO: 13 (the reverse primer),and any functional combination and fragments thereof.

Nucleic acid hybridization reactions are used in various steps describedherein, including association of a labeled probe with the amplified basesequence. The conditions used to achieve a particular level ofstringency will vary depending on the nature of the nucleic acids beinghybridized. For example, the length, degree of complementarity,nucleotide sequence composition (e.g., GC/AT content), and nucleic acidtype (e.g., RNA/DNA) of the hybridizing regions of the nucleic acids canbe considered in selecting hybridization conditions. An example ofprogressively higher stringency conditions is as follows: 2×SSC/0.1% SDS(sodium dodecyl sulfate) at room temperature (hybridization conditions);0.2×SSC/0.1% SDS at room temperature (low stringency conditions);0.2×SSC/0.1% SDS at 42° C. (moderate stringency conditions); and 0.1×SSCat about 68° C. (high stringency conditions).

The kit can include a labeled probe to analyze the amount ofmethylation. The probe of interest can be detectably labeled, forexample, with a radioisotope, a fluorescent compound, a bioluminescentcompound, a chemiluminescent compound, a metal chelator, or an enzyme.Appropriate labeling with such probes is widely known in the art and canbe performed by any conventional method.

The kit can further include instructions for use of the kit to obtain adiagnosis for CS or CLS by compare the amount of methylation of theKILLIN promoter region in the DNA sample to a KILLIN promoter regioncontrol, wherein hypermethylation of the KILLIN promoter regionindicates a diagnosis of Cowden syndrome or Cowden-like Syndrome for thesubject. Instructions included in kits can be affixed to packagingmaterial or can be included as a package insert. While the instructionsare typically written or printed materials they are not limited to such.Any medium capable of storing such instructions and communicating themto an end user is contemplated by this disclosure. Such media include,but are not limited to, electronic storage media (e.g., magnetic discs,tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

Treatment of Cowden and Cowden-Like Syndrome in Subjects Having KILLINPromoter Methylation and/or KILLIN Underexpression

Further embodiments include providing a therapeutic intervention for asubject identified as having a substantially increased risk of havingCowden syndrome or Cowden-like syndrome. The therapeutic invention canbe provided as a follow-up step to a diagnosis of a subject havingCowden syndrome or Cowden-like syndrome, either as a result of carryingout the method of diagnosis described herein or another suitable methodfor diagnosing Cowden syndrome or Cowden-like syndrome. A furtherembodiment provides a method for treating a subject lacks germline PTENmutation and having Cowden syndrome or Cowden-like syndrome. Asdescribed herein, germline PTEN mutation is already known to beassociated with a subset of Cowden syndrome and Cowden-like syndrome.

Therapeutic intervention for Cowden syndrome or Cowden-like syndrome canbe provided to subject by administering to the subject a therapeuticallyeffective amount of a DNA methyltransferase inhibitor. Alternately, orin addition, the method of therapeutic intervention can furtherincluding administering a histone deacetylase inhibitor to the subject.

DNA methyltransferase (DNMT) inhibitors, such as 5-aza-cytidine(5-aza-CR) and 5-aza-2′-deoxycytidine (5-aza-CdR) are widely studiedbecause DNA hypomethylation induces the re-activation of tumorsuppressor genes that are silenced by methylation-mediated mechanisms.The combination of histone deacetylase (HDAC) inhibitors ordemethylating agents with other chemo-therapeutics can be used as apossible molecularly targeted therapeutic strategy. In particular, thecombination of HDAC inhibitors with demethylating agents are effectivesince histones are connected to DNA by both physical and functionalinteractions. As such, the combination of HDAC and DNMT inhibition canbe very effective (and synergistic) in inducing apoptosis,differentiation and/or cell growth arrest in human pancreatic lung,breast, thoracic, leukemia and colon cancer cell lines. Effective agentsinclude HDAC inhibitors, such as, romidepsin, trichostatin A (TSA),sodium butyrate, depsipeptide (FR901228, FK228), valproic acid (VPA) andsuberoylanilide hydroxamic acid (Vorinostat), and the demethylatingagent, 5-aza-CdR used alone and in combination for the treatment ofCowden syndrome or Cowden-like syndrome, or cancer resulting therefrom,in subjects lacking germline PTEN mutation.

The following example is included for purposes of illustration and isnot intended to limit the scope of the invention.

EXAMPLE Example 1 Germline Epigentic Regulation of KILLIN in Cowden andCowden-Like Syndrome

Germline loss-of-function phosphatase and tensin homolog gene (PTEN)mutations cause 25% of Cowden syndrome, a rare autosomal-dominantdisorder (1 in 200,000 live births), characterized by high risks ofbreast, thyroid, and other cancers. A large heterogeneous group ofindividuals with Cowden-like syndrome, who have various combinations ofCowden syndrome features but who do not meet Cowden syndrome diagnosticcriteria, have PTEN mutations less than 10% of the time, makingmolecular diagnosis, prediction, genetic counseling, and risk managementchallenging. Other mechanisms of loss of function such ashypermethylation, which should result in underexpression of PTEN or ofKILLIN, a novel tumor suppressor transcribed in the opposite direction,may account for the remainder of Cowden syndrome and Cowden-likesyndrome. Accordingly, the following experiments were carried out todetermine whether germline methylation is found in Cowden syndrome orCowden-like syndrome in individuals lacking germline PTEN mutations.

Methods Patients

Between October 2005 and December 2009, 2000 patients with Cowdensyndrome or Cowden-like syndrome were prospectively enrolled mainlyregionally and also nationally by the Cleveland Clinic Genomic MedicineInstitute in accordance with research protocol (IRB8458-PTEN) andapproved by the respective institutional review boards for humansubjects protection. All research participants provided written informedconsent. To be enrolled in the IRB8458-PTEN, individuals are eligible ifthey meet the full Cowden syndrome diagnostic criteria established bythe International Cowden Consortium (i.e., major criteria include breastcancer, thyroid cancer, macrocephaly, endometrial carcinoma,Lhermitte-Duclos disease) according to version 2000 (Table 1). Pilarskiet al., J Med Genet., 41(5): 323-326 (2004). Patients meeting therelaxed criteria are referred to as individuals with Cowden-likesyndrome phenotypes (or CSL).

TABLE 1 International Cowden Consortium Operational Diagnostic Criteriafor CowdenSyndrome (2000) Pathognomonic criteria Mucocutaneous lesions:Mucosal lesions Trichilemmomas (facial) Papillomatous lesions Acralkeratoses Major criteria Breast cancer Thyroid cancer, especiallyfollicular thyroid cancer Macrocephaly (occipital frontal circumference97th percentile) Endometrial carcinoma Lhermitte-Duclos disease, definedas presence of a cerebellar dysplastic gangliocytoma Minor criteriaOther thyroid lesions (e.g., goiter) Mental retardation (IQ 75)Hamartomatous intestinal polyps Fibrocystic disease of the breastLipomas Fibromas Genito-urinary tumors (for example, uterine fibroids,renal cell carcinoma) or genito-urinary malformation Operationaldiagnosis if an individual meets any one of the following criteria:Pathognomonic mucocutaneous lesions alone if there are: Six or morefacial papules, of which three or more must be trichilemmoma, orCutaneous facial papules and oral mucosal papillomatosis, or Oralmucosal papillomatosis and acral keratoses, or Six or more palmo plantarkeratoses Two or more major criteria (one must be macrocephaly orLhermitte-Duclos disease) One major and at least three minor criteria Atleast four minor criteria Relatives of individuals with Cowden syndromeare considered to have a diagnosis of CS if they meet any of thefollowing criteria: The pathognomonic criteria Any one major criterionwith or without minor criteria Two minor criteria

Of the 2000 prospectively enrolled participants meeting the criteria forprotocol IRB8458-PTEN, fewer than 400 lacked germline PTEN pathogenicmutations, large deletions, variants of unknown significance, andpolymorphisms by sequencing analysis of all nine exons and the promoter.Of these 400, we selected a nested series of the most recent 123participants who also were found not to have SDHB/D variation,regardless of family history status, comprising 48 with Cowden syndrome,75 with Cowden-like syndrome, and 50 unaffected individuals (populationcontrols resident in the region), for the purposes of this study. Samplesizes were selected to ensure power (P>0.90) to detect a 5% prevalenceof the methylation, as well as to detect a 3-fold difference betweencase and control participants.

All specimens from study and control participants were prepared andanalyzed within the Genomic Medicine Institute. The majority of theparticipants were isolated cases, with the exception of threeindividuals, each of whom had at least one family member who also agreedto be part of our study. All analyses were performed from August 2008through June 2010.

Analysis of Germline Hypermethylation

The combined bisulfite restriction analysis (COBRA; Bennett et al.,Cancer Res.; 67(10): 4657-4664 (2007)) and the bisulfite sequencing wereperformed as previously described. Tada et al., J. Natl Cancer Inst.;98(6): 396-406 (2006). The bisulfite polymerase chain reaction (BS-PCR)primer sequences are shown in Table 2. To provide a comprehensiveanalysis of the methylation status across the CpG islands upstream ofPTEN, we screened four different regions (+400 bp to +700 bp; −188 bp to−477 bp; −425 bp to −640 bp; −806 bp to −1043 bp, all with respect tothe PTEN translation start site).

TABLE 2 Primer Sequences Primer Sequence (5′ to 3′) Tm PTEN_ChIP_FAAAGCTAGCCAGACTCGAGTCAGTGA  55 (SEQ ID NO: 4) PTEN_ChIP_RAAAAGATCTCGAGGCGGACGGGAC  55 (SEQ ID NO: 5) KILLIN_ChIP_FGATGGAAGTTTGAGAGTTGAG  62 (SEQ ID NO: 6) KILLIN_ChIP_RCCACGGCTTCCACCTTCCC  62 (SEQ ID NO: 7) KILLIN_RT_FAAAAGAATTCCGGGGCTGGCGCTTGGGG  60 (SEQ ID NO: 8) KILLIN_RT_RAAAAGCGGCCGCGTCCTTTGGCTTGCTC 60 TTAGG (SEQ ID NO: 9) PTEN_RT_FCAGAAAGACTTGAAGGCGTAT  60 (SEQ ID NO: 10) PTEN_RT_R AACGGCTGAGGGAACTC 60 (SEQ ID NO: 11) BS_PCR_F GTTGTAGTTTTAGGGAGGGGGT  60 (SEQ ID NO: 12)BS_PCR_R CTACTTCTCCTCAACAACCAAAAAC  60 (SEQ ID NO: 13)

Cell Lines, Antibodies, and Plasmids

The patient and control lymphoblastoid cell lines used in this studywere generated from peripheral blood samples by the Genomic MedicineBiorepository of the Cleveland Clinic Genomic Medicine Institute.

Promoter luciferase assay was performed in order to validatetranscriptional repression by DNA promoter methylation and differentialinhibition of TP53 binding. Breast cancer cell line MDA-MB-453 (AmericanType Culture Collection) was used for the luciferase assay.Lymphoblastoid cell lines and MDA-MB-453 (M.D. Anderson metastaticbreast cancer) were maintained in RPMI (Rosewall Park MemorialInstitute) medium with 10% fetal bovine serum and 2% antibiotics. Theantibody used in the chromatin immunoprecipitation analysis (ChIP)experiment was mouse monoclonal TP53 (Santa Cruz; sc-126). The in vitromethylated constructs used for the luciferase assay were generated byfirst digesting 90 μg of the original PTEN and KILLIN promoterconstructs (containing 1 to 1344 bp upstream of the PTEN translationalstart site cloned in either direction) with Bgl II (New England Biolabs,Ipswich, Mass.) and Bbv CI (New England Biolabs). The linearized,digested inserts and vectors were gel extracted. The insert DNA, whichcontain the sequence that is methylated in vivo in patients with Cowdensyndrome or Cowden-like syndrome, was then methylated with CpG Sss Imethylase (New England Biolabs) for 4 hours. Following in vitromethylation, the insert was religated with its corresponding vectorusing a 3:1 insert to vector ratio with 2 μg total DNA. For comparison,the unmethylated counterpart was digested and religated in parallel.

Chromatin Immunoprecipitation Analysis

CUP analysis was performed in order to validate that TP53 binding isdifferentially affected by DNA methylation and performed as previouslydescribed, (Bennett et al., Cancer Res.; 67(10): 4657-4664 (2007))according to the Upstate Cell Signaling Solutions protocol. CUP analysisutilized two controls and four patients that were selected based onmethylation status, representation of Cowden syndrome and Cowden-likesyndrome, and similar levels of KILLIN mRNA down-regulation. Sequencesof the primers used for the quantitative CUP PCRs can be found in Table2.

Luciferase Assays

Luciferase assays were performed as previously described usingMDA-MB-453 cells. Yu et al. Genomics.; 84(4): 647-660 (2004).

Reverse Transcription Polymerase Chain Reaction

The quantitative reverse transcription PCRs were performed as previouslydescribed. Bennett et al., Cancer Res.; 67(10): 4657-4664 (2007). Thestudy population included four controls and eight patients that wereselected based on confirmed methylation status by bisulfite sequencinganalysis and representation of the Cowden syndrome and Cowden-likesyndrome condition.

Demethylation and Histone Deacetylation Inhibition Treatment

The study population included eight patients that were selected based onconfirmed methylation status by bisulfite sequencing analysis andrepresentation of Cowden syndrome and Cowden-like syndrome condition.Demethylation treatment was performed with a cytosine analog,5-aza-2′deoxycytidine (decitabine; Sigma), for 96 hours at 0.5 μMconcentration with approximately 40% confluent suspension lymphoblastoidcells. Inhibition of histone deacetylation was performed with 200 nMconcentration of Trichostatin A (TSA; Sigma) with approximately 40%confluent suspension lymphoblastoid cells for 48 hours, with or without0.5 μM decitabine. The drug was changed daily, and the cells werecollected for RNA isolation.

Statistical Analysis

The statistical significance of the results from reverse transcriptionPCR and luciferase assays was calculated by unpaired t test, with P<0.05being considered statistically significant, using Microsoft Excelversion 12.2.5. The prevalence of component malignancies between KILLINpromoter methylation-positive patients and germline pathogenic PTENmutation-positive patients was compared using the Fisher 2-tailed exacttest with P<0.05 considered to be significant.

Results Germline Methylation in PTEN Mutation-Negative Cowden Syndromeand Cowden-Like Syndrome

We analyzed germline genomic DNA from patients with Cowden syndrome orCowden-like syndrome and from population controls for methylationupstream of PTEN using COBRA. Differential germline methylation wasdetected between 188 and 477 bp upstream of the translation start sitefor PTEN (FIG. 1). All controls showed no methylation (FIG. 2A). Amongthe 123 Cowden syndrome/Cowden-like syndrome samples analyzed, 45 (37%)were hypermethylated compared with all 50 controls (FIG. 2A). Twenty ofthe 48 (42%) classic Cowden syndrome patients without germline PTENmutations showed germline hypermethylation. Of the 75 PTENmutation-negative Cowden-like syndrome patients, 25 (33%) were found tohave germline hypermethylation. Bisulfite sequencing analysis confirmedthese differences in a set of Cowden syndrome and Cowden-like syndromesamples (FIG. 2B).

We then investigated whether methylation segregates with disease infamily members of a proband with germline methylation. Of the 45participants with methylation, only one proband had more than oneaffected family member and more than one unaffected family member whoagreed to enroll in this study. We found germline methylation in four ofsix of the family members, and three of these four had documented Cowdensyndrome/Cowden-like syndrome features (with one unknown phenotype). Thetwo remaining unaffected family members did not have germlinemethylation (30%; 95% confidence interval, 7%-45%; P=0.008).

Germline Methylation and Effect on PTEN and KILLIN Expression

Promoter methylation should result in decreased expression of therelevant gene. In order to validate the pathogenic relevance of thismethylation, the expression of PTEN was analyzed in four control andeight patient cell lines as proof of principle. PTEN expression in themethylated patient samples was surprisingly not decreased, and instead,increased PTEN expression was noted (FIG. 3). The PTEN 5′UTR and codingregion analyzed for methylation overlaps with the putative promoter forKILLIN, a newly characterized tumor suppressor gene (FIG. 1). Cho YJ,Liang P. Proc Natl Acad Sci USA.; 105(14): 5396-5401 (2008). Therefore,in order to address our hypothesis that germline methylation upstream ofPTEN may, instead, be silencing KILLIN, we then analyzed KILLINexpression in the patient samples that showed germline methylation. Inthe methylated patient samples tested, significant underexpression ofKILLIN was observed compared with the control samples (250-fold; 95%confidence interval, 45-14 286; P=0.007) (FIG. 3).

If, in fact, germline methylation down-regulates KILLIN expression, thendemethylation should restore KILLIN expression. DNA methylation andhistone deacetylation of the promoter often work together to achievegene silencing, and histone acetylation has previously been shown to betranscriptionally relevant in the vicinity of the PTEN-KILLINbidirectional promoter. Therefore, we investigated whether reversal ofthese epigenetic modifications, via demethylation and/or inhibition ofhistone deacetylation, would restore only KILLIN expression.KILLIN-methylated patient lymphoblastoid cell lines were treated withthe demethylating drug decitabine and/or the histone deacetylaseinhibitor TSA. Demethylation and/or inhibition of histone deacetylationled to a significant decrease in PTEN expression for seven of the eight(88%) patient cell lines (FIG. 4). In contrast to PTEN, KILLINexpression was restored in 88% (7 of the 8) analyzed patient cell linesfollowing exposure to decitabine and/or TSA (increased 4.88-fold; 95%confidence interval, 1.4-18.1) (FIG. 4).

Germline Methylation Affects TP53 Binding to the KILLIN Promoter

Because the methylation of the shared bidirectional promoter had adifferential impact on transcription for these two genes, we sought tomechanistically explain what might account for the differentialepigenetic control. Both genes are transcriptionally regulated by TP53,and there appears to be two distinct TP53 binding sites—one for KILLINand the other for PTEN. The TP53 binding site for transcriptionalactivation of PTEN lies outside of our germline methylated region(Stambolic et al., Mol Cell. 2001; 8(2): 317-325 (2001)), whereas theputative TP53 binding site for KILLIN lies within the methylated regionidentified in this study (FIG. 5). Therefore, if we are correct that themethylation down-regulates only KILLIN expression, then the methylationshould exclusively inhibit TP53 binding and activation for KILLIN alone,without affecting PTEN transcription (FIG. 5).

One powerful way to interrogate this is by ChIP analysis: if there is nomethylation “blocking” the relevant TP53 binding sites, then CUP shouldreveal the TP53-associated regions of DNA by “pulling down” the sitesbound by TP53 protein via the use of a TP53 antibody. Accordingly, weutilized four lines from patients who exhibited germline methylation ofthe KILLIN promoter and found that in three patients, TP53 bound morestrongly to its PTEN binding site and relatively poorly to its KILLINbinding site, which was blocked by methylation (FIG. 6). As controls,CUP analysis revealed no difference of TP53 binding to both the PTEN andKILLIN TP53 binding sites in the unmethylated control cell lines tested(FIG. 6).

To further address whether the differential TP53 binding of these tworegions is due to methylation seen in the patient samples, weartificially and purposefully methylated the same CpG region in a PTENor KILLIN promoter construct. By overexpressing TP53 in these cells, weobserved a significant increase in PTEN promoter activity, withoutsignificant differences in the level of activation between theunmethylated and methylated PTEN constructs (FIG. 7). Although both theunmethylated and methylated KILLIN constructs also provided an increasein transcriptional activation with TP53 overexpression, the methylatedKILLIN construct showed significantly less transcriptional activation(30%; 95% confidence interval, 7%-45%; P=0.008) by TP53 compared withthe unmethylated KILLIN construct or the PTEN constructs (FIG. 7).

Prevalent Cancers in Pathogenic PTEN Mutation Positive vs KILLINMethylation-Positive Patients

We then turned our attention to the prevalence of component malignanciesin those with germline KILLIN promoter methylation and those with provenpathogenic germline PTEN mutations. We found a significant associationbetween the KILLIN methylation status and prevalence of female breastcancer. In our 42 women with methylation, 35 had invasive breast cancerscompared with 24 of 64 women (from the same IRB8458-PTEN series) withgermline PTEN pathogenic mutations (P<0.0001). Renal cell carcinoma wasoverrepresented in the methylation-positive participants over PTENmutation-positive individuals (4/45 vs 6/155; P=0.004). However, nodifferences in prevalent thyroid cancers or endometrial cancers werefound between the two groups (P=0.2 and 0.4, respectively). Among thetwelve epithelial thyroid carcinomas in individuals with KILLINmethylation-positive Cowden syndrome/Cowden-like syndrome, seven wereclassic papillary thyroid carcinomas compared with the five classicpapillary thyroid carcinomas to ten follicular thyroidcarcinoma/follicular variant of papillary thyroid carcinoma ratio seenin PTEN mutation-positive individuals.

Comments and Conclusions

Individuals with heritable syndromes, such as hereditary nonpolyposiscolorectal cancer, who are negative for mutations in the knownpredisposition genes have rarely been shown to have heritablehypermethylation (also known as epimutation) of the respective promotersof these genes. Hitchins et al., N Engl J Med.; 356(7): 697-705 (2007).This guided our initial hypothesis that a subset of patients with Cowdensyndrome/Cowden-like syndrome without PTEN mutations would possibly havePTEN promoter hypermethylation. Instead, our alternative hypothesis wasproven correct, resulting in our uncovering a novel Cowdensyndrome/Cowden-like syndrome predisposition gene, KILLIN, and a newmechanism of epimutation that contributes to the pathogenesis of Cowdensyndrome/Cowden-like syndrome features in individuals without germlinePTEN mutations. The bidirectional promoter is affected by the distinctmechanism of exclusive disruption of TP53 binding and activation ofKILLIN, while TP53 regulation of PTEN (the latter is outside of themethylated region) remains unaffected.

The germline KILLIN promoter epigenetic modification mechanism describedhere accounts for one-third of germline PTEN mutation-negative Cowdensyndrome and of those whose phenotypic features resemble Cowdensyndrome, prominently those with breast and thyroid disease. In ourcurrent series, more than 40% of PTEN mutation-negative classic Cowdensyndrome and 33% of mutation-negative Cowden-like syndrome patients havegermline epigenetic inactivation of the KILLIN promoter. If these datacan be and must be replicated independently, then a hypothetical schemafor prioritizing gene testing could be as follows: (1) individuals withclassic Cowden syndrome should be offered PTEN testing first; (2) thosefound not to have germline PTEN mutations should then be offered KILLINepigenetic analysis, in the setting of genetic counseling; and (3)individuals with classic Cowden syndrome without germline PTEN mutation(80% are mutation-positive) and without KILLIN epigenetic inactivation(half of the 20% should have KILLIN epigenetic inactivation) should thenbe offered SDHB/D testing (10% of the 20% should have SDHB/D mutation).Altogether, therefore, PTEN, KILLIN, and SDHB/D should then account for92% of all classic Cowden syndrome. Patients with Cowden-like syndromefeatures, especially where breast cancer and/or renal carcinomas arepresent in the individual or family (or both) should be offered KILLINmethylation analysis first because it accounts for 30% of such patientscompared with PTEN mutations, which only account for 5% to 10% of suchindividuals.

By discovering another cancer predisposition gene, we have added to thesensitivity of molecular diagnosis and predictive testing becomespossible. Importantly, genetic counseling and gene-informed riskassessment and management become evidence based. In contrast to germlinePTEN mutations, germline methylation of the KILLIN promoter confers asignificantly higher prevalence of female invasive breast cancer andrenal cell carcinomas. The current national practice guidelines forindividuals with PTEN germline mutations includes heightenedsurveillance of the female breasts and thyroid, but do not haveawareness of renal cancer risk. If our observations of 2- to 3-foldincreased risks of renal and/or breast cancer with KILLIN germlinemethylation over those of PTEN mutation holds, then extra vigilance forthe organs at risk, breast and kidneys, is warranted. TheKILLIN-associated breast cancer risks would parallel those conferred bygermline BRCA1/2 mutations.

Among patients with Cowden syndrome or Cowden-like syndrome, presence ofgermline KILLIN gene promoter hypermethylation was common and wasassociated with increased risk of breast and renal cancer compared withPTEN mutation-positive patients.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1-12. (canceled)
 13. A kit for diagnosing Cowden Syndrome and Cowden-like Syndrome, comprising: a carrier compartmentalized to include a plurality of containers and to receive a DNA sample including the KILLIN promoter region from a subject therein; the carrier comprising a first container including sodium bisulfate, and the solvents and reagents necessary to selectively convert unmethylated cytosine of the DNA sample into uracil; a second container containing a PCR primer pair corresponding to the methylated base sequence of the KILLIN promoter region and the solvents and reagents necessary to obtain an amplified base sequence; a third container containing a labeled probe complementary to the amplified base sequence; and means for detecting the labeled probe to quantitatively analyze the amount of methylation of the KILLIN promoter region; and a KILLIN promoter region control.
 14. The kit of claim 13, wherein the kit further comprises instructions for use of the kit to compare the amount of methylation of the KILLIN promoter region in the DNA sample to a KILLIN promoter region control, wherein hypermethylation of the KILLIN promoter region indicates a diagnosis of Cowden syndrome or Cowden-like Syndrome for the subject.
 15. The kit of claim 13, wherein the PCR primer pair is SEQ ID NO: 12 and SEQ ID NO:
 13. 16. A pair of PCR primer sequences for DNA methylation analysis comprising a forward PCR primer and a reverse PCR primer, wherein the primers include from 20 to 25 nucleotides and are effective to amplify SEQ ID NO: 3 using the polymerase chain reaction.
 17. The PCR primer sequences of claim 16, wherein the forward PCR primer is SEQ ID NO: 12 and the reverse PCR primer is SEQ ID NO:
 13. 18. A method for treating a subject having Cowden syndrome or Cowden-like syndrome by administering to the subject a therapeutically effective amount of a DNA methyltransferase inhibitor.
 19. The method of claim 18, wherein the method further comprises administering a histone deacetylase inhibitor to the subject.
 20. The method of claim 18, wherein the subject has been found to lack germline PTEN mutation.
 21. The method of claim 18, wherein the subject is human. 